Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules

ABSTRACT

Water soluble activated polymers are provided containing an active ethyl sulfone moiety having a reactive site located at the second carbon from the sulfone group. In an embodiment, a poly(ethylene glycol) (PEG) derivative is disclosed that is activated with the active ethyl sulfone moiety for selective attachment to thiol moieties on molecules and surfaces. The activated PEG is water soluble, hydrolytically stable for extended periods, and forms hydrolytically stable linkages with thiol moieties. The linkages generally are not reversible in reducing environments. The PEG derivative is useful for modifying the characteristics of substances including modifying biologically active molecules and surfaces for biocompatibility.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 10/647,621,filed Aug. 25, 2003, now U.S. Pat. No. 6,894,025, which is acontinuation or application Ser. No. 09/294,188, filed Apr. 19, 1999,now U.S. Pat. No. 6,610,281, which is a divisional of application Ser.No. 09/027,679, filed Feb. 23, 1998, now U.S. Pat. No. 5,900,461, whichis a divisional of application Ser. No. 08/473,734, filed Jun. 7, 1995,now U.S. Pat. No. 5,739,208, which is a divisional of application Ser.No. 08/151,481, filed Nov. 12, 1993, now U.S. Pat. No. 5,446,090, all ofwhich are incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to active derivatives of poly(ethylene glycol)and related hydrophilic polymers and to methods for their synthesis foruse in modifying the characteristics of surfaces and molecules.

BACKGROUND OF THE INVENTION

Poly(ethylene glycol) (“PEG”) has been studied for use inpharmaceuticals, on artificial implants, and in other applications wherebiocompatibility is of importance. Various derivatives of poly(ethyleneglycol) (“PEG derivatives”) have been proposed that have an activemoiety for permitting PEG to be attached to pharmaceuticals and implantsand to molecules and surfaces generally to modify the physical orchemical characteristics of the molecule or surface.

For example, PEG derivatives have been proposed for coupling PEG tosurfaces to control wetting, static buildup, and attachment of othertypes of molecules to the surface, including proteins or proteinresidues. More specifically, PEG derivatives have been proposed forattachment to the surfaces of plastic contact lenses to reduce thebuildup of proteins and clouding of vision. PEG derivatives have beenproposed for attachment to artificial blood vessels to reduce proteinbuildup and the danger of blockage. PEG derivatives have been proposedfor immobilizing proteins on a surface, as in enzymatic catalysis ofchemical reactions.

In still further examples, PEG derivatives have been proposed forattachment to molecules, including proteins, for protecting the moleculefrom chemical attack, to limit adverse side effects of the molecule, orto increase the size of the molecule, thereby potentially to renderuseful substances that have some medicinal benefit, but are otherwisenot useful or are even harmful to a living organism. Small moleculesthat normally would be excreted through the kidneys are maintained inthe blood stream if their size is increased by attaching a biocompatiblePEG derivative. Proteins and other substances that create an immuneresponse when injected can be hidden to some degree from the immunesystem by coupling of a PEG molecule to the protein.

PEG derivatives have also been proposed for affinity partitioning of,for example, enzymes from a cellular mass. In affinity partitioning, thePEG derivative includes a functional group for reversible coupling to anenzyme that is contained within a cellular mass. The PEG and enzymeconjugate is separated from the cellular mass and then the enzyme isseparated from the PEG derivative, if desired.

Coupling of PEG derivatives to proteins illustrates some of the problemsthat have been encountered in attaching PEG to surfaces and molecules.For many surfaces and molecules, the number of sites available forcoupling reactions with a PEG derivative is somewhat limited. Forexample, proteins typically have a limited number and distinct type ofreactive sites available for coupling. Even more problematic, some ofthe reactive sites may be responsible for the protein's biologicalactivity, as when an enzyme catalyzes certain chemical reactions. A PEGderivative that attached to a sufficient number of such sites couldadversely affect the activity of the protein.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are built of various sequences of alpha-amino acids, which havethe general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—) of oneamino acid joins to the carboxyl moiety (—COOH) of an adjacent aminoacid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where _(n) can be hundreds or thousands. The fragmentrepresented by R can contain reactive sites for protein biologicalactivity and for attachment of PEG derivatives.

For example, in lysine, which is an amino acid forming part of thebackbone of most proteins, an —NH₂ moiety is present in the epsilonposition as well as in the alpha position. The epsilon —NH₂ is free forreaction under conditions of basic pH. Much of the art has been directedto developing PEG derivatives for attachment to the epsilon —NH₂ moietyof the lysine fraction of a protein. These PEG derivatives all have incommon that the lysine amino acid fraction of the protein typically isinactivated, which can be a drawback where lysine is important toprotein activity.

Zalipsky U.S. Pat. No. 5,122,614 discloses that PEG molecules activatedwith an oxycarbonyl-N-dicarboximide functional group can be attachedunder aqueous, basic conditions by a urethane linkage to the amine groupof a polypeptide. Activated PEG-N-succinimide carbonate is said to formstable, hydrolysis-resistant urethane linkages with amine groups. Theamine group is shown to be more reactive at basic pHs of from about 8.0to 9.5, and reactivity falls off sharply at lower pH. However,hydrolysis of the uncoupled PEG derivative also increases sharply at pHsof 8.0 to 9.5. Zalipsky avoids the problem of an increase in the rate ofreaction of the uncoupled PEG derivative with water by using an excessof PEG derivative to bind to the protein surface. By using an excess,sufficient reactive epsilon amino sites are bound with PEG to modify theprotein before the PEG derivative has an opportunity to becomehydrolyzed and unreactive.

Zalipsky's method is adequate for attachment of the lysine fraction of aprotein to a PEG derivative at one active site on the PEG derivative.However, if the rate of hydrolysis of the PEG derivative is substantial,then it can be problematic to provide attachment at more than one activesite on the PEG molecule, since a simple excess does not slow the rateof hydrolysis.

For example, a linear PEG with active sites at each end will attach to aprotein at one end, but, if the rate of hydrolysis is significant, willreact with water at the other end to become capped with a relativelynonreactive hydroxyl moiety, represented structurally as —OH, ratherthan forming a “dumbbell” molecular structure with attached proteins orother desirable groups on each end. A similar problem arises if it isdesired to couple a molecule to a surface by a PEG linking agent becausethe PEG is first attached to the surface or couples to the molecule, andthe opposite end of the PEG derivative must remain active for asubsequent reaction. If hydrolysis is a problem, then the opposite endtypically becomes inactivated.

Also disclosed in Zalipsky U.S. Pat. No. 5,122,614 are several other PEGderivatives from prior patents. PEG-succinoyl-N-hydroxysuccinimide esteris said to form ester linkages that have limited stability in aqueousmedia, thus indicating an undesirable short half-life for thisderivative. PEG-cyanuric chloride is said to exhibit an undesirabletoxicity and to be non-specific for reaction with particular functionalgroups on a protein. The PEG-cyanuric chloride derivative may thereforehave undesirable side effects and may reduce protein activity because itattaches to a number of different types of amino acids at variousreactive sites. PEG-phenylcarbonate is said to produce toxic hydrophobicphenol residues that have affinity for proteins. PEG activated withcarbonyldiimidazole is said to be too slow in reacting with proteinfunctional groups, requiring long reaction times to obtain sufficientmodification of the protein.

Still other PEG derivatives have been proposed for attachment tofunctional groups on amino acids other than the epsilon —NH₂ of lysine.Histidine contains a reactive imino moiety, represented structurally as—N(H)—, but many derivatives that react with epsilon —NH₂ also reactwith —N(H)—. Cysteine contains a reactive thiol moiety, representedstructurally as —SH, but the PEG derivative maleimide that is reactivewith this moiety is subject to hydrolysis.

As can be seen from the small sampling above, considerable effort hasgone into developing various PEG derivatives for attachment to, inparticular, the —NH₂ moiety on the lysine amino acid fraction of variousproteins. Many of these derivatives have proven problematic in theirsynthesis and use. Some form unstable linkages with the protein that aresubject to hydrolysis and therefore do not last very long in aqueousenvironments, such as in the blood stream. Some form more stablelinkages, but are subject to hydrolysis before the linkage is formed,which means that the reactive group on the PEG derivative may beinactivated before the protein can be attached. Some are somewhat toxicand are therefore less suitable for use in vivo. Some are too slow toreact to be practically useful. Some result in a loss of proteinactivity by attaching to sites responsible for the protein's activity.Some are not specific in the sites to which they will attach, which canalso result in a loss of desirable activity and in a lack ofreproducibility of results.

SUMMARY OF THE INVENTION

The invention provides water soluble and hydrolytically stablederivatives of poly(ethylene glycol) (“PEG”) polymers and relatedhydrophilic polymers having one or more active sulfone moieties. Thesepolymer derivatives with active sulfone moieties are highly selectivefor coupling with thiol moieties instead of amino moieties on moleculesand on surfaces, especially at pHs of about 9 or less. The sulfonemoiety, the linkage between the polymer and the sulfone moiety, and thelinkage between the thiol moiety and the sulfone moiety are notgenerally reversible in reducing environments and are stable againsthydrolysis for extended periods in aqueous environments at pHs of about11 or less. Consequently, the physical and chemical characteristics of awide variety of substances can be modified under demanding aqueousconditions with the active sulfone polymer derivatives. For example,conditions for modification of biologically active substances can beoptimized to preserve a high degree of biological activity.Pharmaceuticals from aspirin to penicillin can be usefully modified byattachment of active sulfone polymer derivatives if thesepharmaceuticals are modified to contain thiol moieties. Large proteinscontaining cysteine units, which have active thiol moieties, can also beusefully modified. Techniques of recombinant DNA technology (“geneticengineering”) can be used to introduce cysteine groups into desiredplaces in a protein. These cysteines can be coupled to active sulfonepolymer derivatives to provide hydrolytically stable linkages on avariety of proteins that do not normally contain cysteine units.

Specific sulfone moieties for the activated polymers of the inventionare those having at least two carbon atoms joined to the sulfone group—SO₂— with a reactive site for thiol specific coupling reactions on thesecond carbon from the sulfone group.

More specifically, the active sulfone moieties comprise vinyl sulfone,the active ethyl sulfones, including the haloethyl sulfones, and thethiol-specific active derivatives of these sulfones. The vinyl sulfonemoiety can be represented structurally as —SO₂—CH═CH₂; the active ethylsulfone moiety can be represented structurally as —SO₂— CH₂—CH₂—Z, whereZ can be halogen or some other leaving group capable of substitution bythiol to form the sulfone and thiol linkage —SO₂—CH₂—CH₂—S—W, where Wrepresents a biologically active molecule, a surface, or some othersubstance. The derivatives of the vinyl and ethyl sulfones can includeother substituents, so long as the water solubility and thethiol-specific reactivity of the reactive site on the second carbon aremaintained.

The invention includes hydrolytically stable conjugates of substanceshaving thiol moieties with polymer derivatives having active sulfonemoieties. For example, a water soluble sulfone-activated PEG polymer canbe coupled to a biologically active molecule at a reactive thiol site.The linkage by which the PEG and the biologically active molecule arecoupled includes a sulfone moiety coupled to a thiol moiety and has thestructure PEG—SO₂—CH₂—CH₂—S—W, where W represents the biologicallyactive molecule, whether the sulfone moiety prior to coupling of the PEGwas vinyl sulfone or an active ethyl sulfone.

The invention also includes biomaterials comprising a surface having oneor more reactive thiol sites and one or more of the water solublesulfone-activated polymers of the invention coupled to the surface by asulfone and thiol linkage. Biomaterials and other substances can also becoupled to the sulfone activated polymer derivatives through a linkageother than the sulfone and thiol linkage, such as a conventional aminolinkage, to leave a more hydrolytically stable activating group, thesulfone moiety, available for subsequent reactions.

The invention includes a method of synthesizing the activated polymersof the invention. A sulfur containing moiety is bonded directly to acarbon atom of the polymer and then converted to the active sulfonemoiety. Alternatively, the sulfone moiety can be prepared by attaching alinking agent that has the sulfone moiety at one terminus to aconventional activated polymer so that the resulting polymer has thesulfone moiety at its terminus.

More specifically, a water soluble polymer having at least one activehydroxyl moiety undergoes a reaction to produce a substituted polymerhaving a more reactive moiety thereon. The resulting substituted polymerundergoes a reaction to substitute for the more reactive moiety asulfur-containing moiety having at least two carbon atoms where thesulfur in the sulfur-containing moiety is bonded directly to a carbonatom of the polymer. The sulfur-containing moiety then undergoesreactions to oxidize sulfur, —S—, to sulfone, —SO₂—, and to provide asufficiently reactive site on the second carbon atom of the sulfonecontaining moiety for formation of linkages with thiol containingmoieties.

Still more specifically, the method of synthesizing the activatedpolymers of the invention comprises reacting poly(ethylene glycol) witha hydroxyl activating compound to form an ester or with a halogencontaining derivative to form a halogen substituted PEG. The resultingactivated PEG is then reacted with mercaptoethanol to substitute themercaptoethanol radical for the ester moiety or the halide. The sulfurin the mercaptoethanol moiety is oxidized to sulfone. The ethanolsulfone is activated by either activating the hydroxyl moiety orsubstituting the hydroxyl moiety with a more active moiety such ashalogen. The active ethyl sulfone of PEG can then be converted to vinylsulfone, if desired, by cleaving the activated hydroxyl or other activemoiety and introducing the carbon-carbon double bond adjacent thesulfone group —SO₂—.

The invention also includes a method for preparing a conjugate of asubstance with a polymer derivative that has an active sulfone moiety.The method includes the step of forming a linkage between the polymerderivative and the substance, which linkage can be between the sulfonemoiety and a thiol moiety.

Thus the invention provides activated polymers that are specific inreactivity, stable in water, stable in reducing environments, and thatform more stable linkages with surfaces and molecules, includingbiologically active molecules, than previously has been achieved. Theactivated polymer can be used to modify the characteristics of surfacesand molecules where biocompatibility is of importance. Because theactivated polymer is stable under aqueous conditions and forms stablelinkages with thiol moieties, the most favorable reaction conditions canbe selected for preserving activity of biologically active substancesand for optimizing the rate of reaction for polymer coupling.

DETAILED DESCRIPTION OF THE INVENTION

The synthetic route used to prepare active sulfones of poly(ethyleneglycol) and related polymers comprises at least four steps in whichsulfur is bound to a polymer molecule and then converted through aseries of reactions to an active sulfone functional group. The startingPEG polymer molecule has at least one hydroxyl moiety, —OH, that isavailable to participate in chemical reactions and is considered to bean “active” hydroxyl moiety. The PEG molecule can have multiple activehydroxyl moieties available for chemical reaction, as is explainedbelow. These active hydroxyl moieties are in fact relativelynonreactive, and the first step in the synthesis is to prepare a PEGhaving a more reactive moiety.

A more reactive moiety typically will be created by one of two routes,hydroxyl activation or substitution. Other methods are available asshould be apparent to the skilled artisan, but hydroxyl activation andsubstitution are the two most often used. In hydroxyl activation, thehydrogen atom —H on the hydroxyl moiety —OH is replaced with a moreactive group. Typically, an acid or an acid derivative such as an acidhalide is reacted with the PEG to form a reactive ester in which the PEGand the acid moiety are linked through the ester linkage. The acidmoiety generally is more reactive than the hydroxyl moiety. Typicalesters are the sulfonate, carboxylate, and phosphate esters.

Sulfonyl acid halides that are suitable for use in practicing theinvention include methanesulfonyl chloride and p-toluenesulfonylchloride. Methanesulfonyl chloride is represented structurally asCH₃SO₂Cl and is also known as mesyl chloride. Methanesulfonyl esters aresometimes referred to as mesylates. Para-toluenesulfonyl chloride isrepresented structurally as H₃CC₆H₄SO₂Cl and is also known as tosylchloride. Toluenesulfonyl esters are sometimes referred to as tosylates.

In a substitution reaction, the entire —OH group on the PEG issubstituted by a more reactive moiety, typically a halide. For example,thionyl chloride, represented structurally as SOCl₂, can be reacted withPEG to form a more reactive chlorine substituted PEG. Substitution ofthe hydroxyl moiety by another moiety is sometimes referred to in theart as hydroxyl activation. The term “hydroxyl activation” should beinterpreted herein to mean substitution as well as esterification andother methods of hydroxyl activation.

The terms “group,” “functional group,” “moiety,” “active moiety,”“reactive site,” and “radical” are somewhat synonymous in the chemicalarts and are used in the art and herein to refer to distinct, definableportions or units of a molecule and to units that perform some functionor activity and are reactive with other molecules or portions ofmolecules. In this sense a protein or a protein residue can beconsidered a molecule or as a functional group or moiety when coupled toa polymer.

The term “PEG” is used in the art and herein to describe any of severalcondensation polymers of ethylene glycol having the general formularepresented by the structure H(OCH₂CH₂)_(n)OH. PEG is also known aspolyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol.PEG can be prepared as copolymers of ethylene oxide and many othermonomers.

Poly(ethylene glycol) is used in biological applications because it hasproperties that are highly desirable and is generally approved forbiological or biotechnical applications. PEG typically is clear,colorless, odorless, soluble in water, stable to heat, inert to manychemical agents, does not hydrolyze or deteriorate, and is nontoxic.Poly(ethylene glycol) is considered to be biocompatible, which is to saythat PEG is capable of coexistence with living tissues or organismswithout causing harm. More specifically, PEG is not immunogenic, whichis to say that PEG does not tend to produce an immune response in thebody. When attached to a moiety having some desirable function in thebody, the PEG tends to mask the moiety and can reduce or eliminate anyimmune response so that an organism can tolerate the presence of themoiety. Accordingly, the sulfone-activated PEGs of the invention shouldbe substantially non-toxic and should not tend substantially to producean immune response or cause clotting or other undesirable effects.

The second step in the synthesis is to link sulfur directly to a carbonatom in the polymer and in a form that can be converted to an ethylsulfone or ethyl sulfone derivative having similar reactive properties.“Ethyl” refers to a moiety having an identifiable group of two carbonatoms joined together. The active sulfone PEG derivative requires thatthe second carbon atom in the chain away from the sulfone group providea reactive site for linkages of thiol moieties with the sulfone. Thisresult can be achieved by reacting the active moiety produced in thefirst step mentioned above, which typically will be the ester or halidesubstituted PEG, in a substitution reaction with an alcohol that alsocontains a reactive thiol moiety attached to an ethyl group, athioethanol moiety. The thiol moiety is oxidized to sulfone and thesecond carbon away from the sulfone on the ethyl group is converted to areactive site.

Compounds containing thiol moieties, —SH, are organic compounds thatresemble alcohols, which contain the hydroxyl moiety —OH, except that inthiols, the oxygen of at least one hydroxyl moiety is replaced bysulfur. The activating moiety on the PEG derivative from the firstreaction, which typically is either halide or the acid moiety of anester, is cleaved from the polymer and is replaced by the alcoholradical of the thioethanol compound. The sulfur in the thiol moiety ofthe alcohol is linked directly to a carbon on the polymer.

The alcohol should be one that provides a thioethanol moiety forattachment directly to the carbon of the polymer chain, or that caneasily be converted to a thioethanol moiety or substituted moiety ofsimilar reactive properties. An example of such an alcohol ismercaptoethanol, which is represented structurally as HSCH₂CH₂OH and issometimes also called thioethanol.

In the third step of the synthesis, an oxidizing agent is used toconvert the sulfur that is attached to the carbon to the sulfone group,—SO₂. There are many such oxidizing agents, including hydrogen peroxideand odium perborate. A catalyst, such as tungstic acid, can be useful.However, the sulfone that is formed is not in a form active forthiol-selective reactions and it is necessary to remove the relativelyunreactive hydroxyl moiety of the alcohol that was added in thesubstitution reaction of the second step.

In the fourth step, the hydroxyl moiety of the alcohol that was added inthe second step is converted to a more reactive form, either throughactivation of the hydroxyl group or through substitution of the hydroxylgroup with a more reactive group, similar to the first step in thereaction sequence. Substitution typically is with halide to form ahaloethyl sulfone or a derivative thereof having a reactive site on thesecond carbon removed from the sulfone moiety. Typically, the secondcarbon on the ethyl group will be activated by a chloride or bromidehalogen. Hydroxyl activation should provide a site of similarreactivity, such as the sulfonate ester. Suitable reactants are theacids, acid halides, and others previously mentioned in connection withthe first step in the reaction, especially thionyl chloride forsubstitution of the hydroxyl group with the chlorine atom.

The resulting polymeric activated ethyl sulfone is stable, isolatable,and suitable for thiol-selective coupling reactions. As shown in theexamples, PEG chloroethyl sulfone is stable in water at a pH of about 7or less, but nevertheless can be used to advantage for thiol-selectivecoupling reactions at conditions of basic pH up to at least about pH 9.

In the thiol coupling reaction, it is possible that the thiol moietydisplaces chloride, as in the following reaction:PEG—SO₂—CH₂—CH₂—Cl+W—S—H→PEG—SO₂—CH₂—CH₂—S—W,where W represents the moiety to which the thiol moiety SH is linked andcan be a biologically active molecule, a surface, or some othersubstance. However, and while not wishing to be bound by theory, it isbelieved, based on the observable reaction kinetics as shown in Example3, that the chloroethyl and other activated ethyl sulfones and reactivederivatives are converted to PEG vinyl sulfone, and that it is the PEGvinyl sulfone or derivative thereof that is actually linked to the thiolmoiety. Nevertheless, the resulting sulfone and thiol linkage is notdistinguishable, whether from active PEG ethyl sulfone or from PEG vinylsulfone, and so the active ethyl sulfone can be used at pHs above 7 forlinking to thiol groups.

PEG vinyl sulfone is also stable and isolatable and can formthiol-selective, hydrolytically stable linkages, typically in much lesstime than the haloethyl sulfone or other activated ethyl sulfone, asexplained further below.

In a fifth step that can be added to the synthesis, the activated ethylsulfone is reacted with any of a variety of bases, such as sodiumhydroxide or triethylamine, to form PEG vinyl sulfone or one of itsactive derivatives for use in thiol-selective coupling reactions.

As shown in the examples below, especially Example 3, PEG vinyl sulfonereacts quickly with thiol moieties and is stable against hydrolysis inwater of pH less than about 11 for at least several days. The reactioncan be represented as follows:PEG—SO₂—CH═CH₂+W—S—H→PEG—SO₂—CH₂—CH₂—S—W.The thiol moiety is said to add “across the double bond.” The W—S moietyadds to the terminal CH₂ of the double bond, which is the second carbonfrom the sulfone group SO₂. The hydrogen H adds to the CH of the doublebond. However, at a pH of above about 9, selectivity of the sulfonemoiety for thiol is diminished and the sulfone moiety becomes somewhatmore reactive with amino groups.

Alternatively to the above synthesis, the sulfone-activated PEGderivatives can be prepared by attaching a linking agent having asulfone moiety to a PEG activated with a different functional group. Forexample, an amino activated PEG, PEG—NH₂, is reacted under favorableconditions of pH of about 9 or less with a small molecule that has asuccinimidyl active ester moiety NHS—O₂C— at one terminus and a sulfonemoiety, vinyl sulfone —SO₂—CH═CH₂, at the other terminus. The aminoactivated PEG forms a stable linkage with the succinimidyl ester. Theresulting PEG is activated with the vinyl sulfone moiety at the terminusand is hydrolytically stable. The reaction and the resulting vinylsulfone activated PEG are represented structurally as follows:PEG—NH₂+NHS—O₂C—CH₂—CH₂—SO₂—CH═CH₂→PEG—NH—OC—CH₂—CH₂—SO₂—CH═CH₂.

A similar activated PEG could be achieved by reacting an amine-activatedPEG such as succinimidyl active ester PEG, PEG—CO₂—NHS, with a smallmolecule that has an amine moiety at one terminus and a vinyl sulfonemoiety at the other terminus. The succinimidyl ester forms a stablelinkage with the amine moiety as follows:PEG—CO₂—NHS+NH₂—CH₂—CH₂—SO₂—CH═CH₂→PEG—CO—NH—CH₂—CH₂—SO₂—CH═CH₂.

The active PEG sulfones of the invention can be of any molecular weightand can be linear or branched with hundreds of arms. The PEG can besubstituted or unsubstituted so long as at least one reactive site isavailable for substitution with the sulfone moieties. PEG typically hasaverage molecular weights of from 200 to 100,000 and its biologicalproperties can vary with molecular weight and depending on the degree ofbranching and substitution, so not all of these derivatives may beuseful for biological or biotechnical applications. For most biologicaland biotechnical applications, substantially linear, straight-chain PEGvinyl sulfone or bis vinyl sulfone or activated ethyl sulfone will beused, substantially unsubstituted except for the vinyl sulfone or ethylsulfone moieties and, where desired, other additional functional groups.For many biological and biotechnical applications, the substituentswould typically be unreactive groups such as hydrogen H— and methyl CH₃—(“m-PEG”).

The PEG can have more than one vinyl sulfone or precursor moietyattached or the PEG can be capped on one end with a relativelynonreactive moiety such as the methyl radical, —CH₃. The capped form canbe useful, for example, if it is desirable simply to attach the polymerchains at various thiol sites along a protein chain. Attachment of PEGmolecules to a biologically active molecule such as a protein or otherpharmaceutical or to a surface is sometimes referred to as “PEGylation.”

A linear PEG with active hydroxyls at each end can be activated at eachend with vinyl sulfone or its precursor or derivatives of similarreactivity to become bifunctional. The bifunctional structure, PEG bisvinyl sulfone, for example, is sometimes referred to as a dumbbellstructure and can be used, for example, as a linker or spacer to attacha biologically active molecule to a surface or to attach more than onesuch biologically active molecule to the PEG molecule. The stability ofthe sulfone moiety against hydrolysis makes it particularly useful forbifunctional or heterobifunctional applications.

Another application for PEG vinyl sulfone and its precursor is dendriticactivated PEG in which multiple arms of PEG are attached to a centralcore structure. Dendritic PEG structures can be highly branched and arecommonly known as “star” molecules. Star molecules are generallydescribed in Merrill U.S. Pat. No. 5,171,264, the contents of which areincorporated herein by reference. The sulfone moieties can be used toprovide an active, functional group on the end of the PEG chainextending from the core and as a linker for joining a functional groupto the star molecule arms.

PEG vinyl sulfone and its precursors and derivatives can be used forattachment directly to surfaces and molecules having a thiol moiety.However, more typically a heterobifunctional PEG derivative having asulfone moiety on one terminus and a different functional moiety on theopposite terminus group will be attached by the different moiety to asurface or molecule. When substituted with one of the other activemoieties, the heterobifunctional PEG dumbbell structure can be used, forexample, to carry a protein or other biologically active molecule bysulfone linkages on one end and by another linkage on the other end,such as an amine linkage, to produce a molecule having two differentactivities. A heterobifunctional PEG having a sulfone moiety on one endand an amine specific moiety on the other end could be attached to bothcysteine and lysine fractions of proteins. A stable amine linkage can beachieved and then the hydrolytically stable unreacted sulfone moiety isavailable for subsequent thiol-specific reactions as desired.

Other active groups for heterobifunctional sulfone-activated PEGs can beselected from among a wide variety of compounds. For biological andbiotechnical applications, the substituents would typically be selectedfrom reactive moieties typically used in PEG chemistry to activate PEGsuch as the aldehydes, trifluoroethylsulfonate, which is also sometimescalled tresylate, n-hydroxylsuccinimide ester, cyanuric chloride,cyanuric fluoride, acyl azide, succinate, the p-diazo benzyl group, the3-(p-diazophenyloxy)-2-hydroxy propyloxy group, and others.

Examples of active moieties other than sulfone are shown in Davis et al.U.S. Pat. No. 4,179,337; Lee et al. U.S. Pat. Nos. 4,296,097 and4,430,260; Iwasaki et al. U.S. Pat. No. 4,670,417; Katre et al. U.S.Pat. Nos. 4,766,106; 4,917,888; and 4,931,544; Nakagawa et al. U.S. Pat.No. 4,791,192; Nitecki et al. U.S. Pat. Nos. 4,902,502 and 5,089,261;Saifer U.S. Pat. No. 5,080,891; Zalipsky U.S. Pat. No. 5,122,614; Shadleet al. U.S. Pat. No. 5,153,265; Rhee et al. U.S. Pat. No. 5,162,430;European Patent Application Publication No. 0 247 860; and PCTInternational Application Nos. US86/01252; GB89/01261; GB89/01262;GB89/01263; US90/03252; US90/06843; US91/06103; US92/00432; andUS92/02047, the contents of which are incorporated herein by reference.

It should be apparent to the skilled artisan that the dumbbellstructures discussed above could be used to carry a wide variety ofsubstituents and combinations of substituents. Pharmaceuticals such asaspirin, vitamins, penicillin, and others too numerous to mention;polypeptides or proteins and protein fragments of variousfunctionalities and molecular weights; cells of various types; surfacesfor biomaterials, almost any substance could be modified. As usedherein, the term “protein” should be understood to include peptides andpolypeptides, which are polymers of amino acids. The term “biomaterial”means a material, typically synthetic and sometimes made of plastic,that is suitable for implanting in a living body to repair damaged ordiseased parts. An example of a biomaterial is artificial blood vessels.

One straight chain PEG derivative of the invention for biological andbiotechnical applications has the basic structure R—(OCH₂CH₂)_(n)—Y. ThePEG monomer OCH₂CH₂ preferably is substantially unsubstituted andunbranched along the polymer backbone. The subscript “_(n)” can equalfrom about 5 to 3,000. A more typical range is from about 5 to 2,200,which corresponds to a molecular weight of from about 220 to 100,000.Still more typical is a range of from about 34 to 1,100, whichcorresponds to a molecular weight range of from about 1,500 to 50,000.Most applications will be accomplished with molecular weights in theneighborhood of 2,000 to 5,000, which corresponds to a value of _(n) offrom about 45 to 110.

In the above structure, Y represents —SO₂—CH═CH₂ or —SO₂—CH₂—CH₂—X whereX is a halogen. R represents a group that may be the same or differentfrom Y. R can be H—, H₃C—, CH₂═CH—SO₂—, Cl—CH₂—CH₂—SO₂—, or a polymeractivating group other than CH₂═CH—SO₂—, Cl—CH₂—CH₂—SO₂—, such as isreferred to with respect to the above patents and published patentapplications.

The active polymer derivatives are water soluble and hydrolyticallystable and produce water soluble and hydrolytically stable linkages withthiol groups. The derivatives are considered infinitely soluble in wateror as approaching infinite solubility and can enable otherwise insolublemolecules to pass into solution when conjugated with the derivative.

Hydrolytic stability of the derivatives means that the linkage betweenthe polymer and the sulfone moiety is stable in water and that the vinylsulfone moiety does not react with water at a pH of less than about 11for an extended period of time of at least several days, and potentiallyindefinitely, as shown in Example 3 below. The activated ethyl sulfonecan be converted to the vinyl sulfone at conditions of basic pH, withthe same resulting stability. Hydrolytic stability of the thiol linkagemeans that conjugates of the activated polymer and a substance having athiol moiety are stable at the sulfone-thiol linkage for an extendedperiod of time in aqueous environments at a pH of below about 11. Mostproteins could be expected to lose their activity at a caustic pH of 11or higher, so it should be apparent to the skilled artisan that manyapplications for the active sulfone PEG derivatives will be at pHs ofless than 11, regardless of the stability of the sulfone moiety athigher pH.

To be useful for modification of proteins and other substances, it isonly necessary that the sulfone be stable for a period of timesufficient to permit the sulfone to react with a reactive thiol moietyon the protein or other substance. The rate of reaction of the sulfonemoiety with thiol can vary with pH, as shown in Example 2 below, fromabout 2 minutes to 30 minutes, which is much faster than the rate ofhydrolysis, if any. Vinyl sulfone could be expected to react with thiolover a much broader range of reaction times since it is stable for longperiods of time. Also, as shown in Example 3 below, at conditions ofbasic pH chloroethyl sulfone is not hydrolyzed, but is converted tovinyl sulfone, which remains stable for several days and is even morereactive toward thiol groups. Accordingly, for the purpose of modifyingthe characteristics of substances, the active ethyl sulfones can also beconsidered to be hydrolytically stable for an extended period of timeover a broad pH range.

Other water soluble polymers than PEG are believed to be suitable forsimilar modification and activation with an active sulfone moiety. Theseother polymers include poly(vinyl alcohol) (“PVA”); other poly(alkyleneoxides) such as poly(propylene glycol) (“PPG”) and the like; andpoly(oxyethylated polyols) such as poly(oxyethylated glycerol),poly(oxyethylated sorbitol), and poly(oxyethylated glucose), and thelike. The polymers can be homopolymers or random or block copolymers andterpolymers based on the monomers of the above polymers, straight chainor branched, or substituted or unsubstituted similar to PEG, but havingat least one active site available for reaction to form the sulfonemoiety.

The following Example 1 shows the synthesis, isolation, andcharacterization of poly(ethylene glycol) chloroethyl sulfone followedby the preparation of poly(ethylene glycol) vinyl sulfone from thechloroethyl sulfone. Preparation of other polymeric sulfones having areactive site on the second carbon from the sulfone group is similar andthe steps for doing so should be apparent to the skilled artisan basedon Example 1 below and the polymers listed above.

EXAMPLE 1

Synthesis

The reaction steps can be illustrated structurally as follows:

-   (1) PEG—OH+CH₃SO₂Cl→PEG—OSO₂CH₃-   (2) PEG—OSO₂CH₃+HSCH₂CH₂OH→PEG—SCH₂CH₂OH-   (3) PEG—SCH₂CH₂OH+H₂O₂→PEG—SO₂CH₂CH₂OH-   (4) PEG—SO₂CH₂CH₂OH+SOCl₂→PEG—SO₂CH₂CH₂Cl-   (5) PEG—SO₂—CH₂CH₂Cl+NaOH→PEG—SO₂—CH═CH₂+HCl

Each of the above reactions is described in detail below:

Reaction 1. Reaction 1 represents the preparation of the methanesulfonyl ester of poly(ethylene glycol), which can also be referred toas the methanesulfonate or mesylate of poly(ethylene glycol). Thetosylate and the halides can be prepared by similar procedures, whichare believed to be apparent to the skilled artisan.

To prepare the mesylate, twenty-five grams of PEG of molecular weight3400 was dried by azeotropic distillation in 150 ml of toluene.Approximately half of the toluene was distilled off in drying the PEG.Forty ml of dry dichloromethane was added to the toluene and PEGsolution, followed by cooling in an ice bath. To the cooled solution wasadded 1.230 ml of distilled methanesulfonyl chloride, which is anequivalent weight of 1.06 with respect to PEG hydroxyl groups, and 2.664ml of dry triethylamine, which is an equivalent weight of 1.3 withrespect to PEG hydroxyl groups. “Equivalent weight” as used above can bethought of as “combining weight” and refers to the weight of a compoundthat will react with an equivalent weight of PEG hydroxyl groups.

The reaction was permitted to sit overnight during which time it warmedto room temperature. Triethylammonium hydrochloride precipitated and theprecipitate was removed by filtration. Thereafter, the volume wasreduced by rotary evaporation to 20 ml. The mesylate was precipitated byaddition to 100 ml of cold dry ethyl ether. Nuclear magnetic resonance(NMR) analysis showed 100% conversion of hydroxyl groups to mesylategroups.

Reaction 2. Reaction 2 represents the formation of poly(ethylene glycol)mercaptoethanol by reaction of the mesylate with mercaptoethanol. Thereaction causes the methanesulfonate radical to be displaced from thePEG. The sulfur in the mercaptoethanol radical is attached directly tothe carbon in the carbon-carbon backbone of the PEG.

Twenty grams of the mesylate from reaction 1 was dissolved in 150 ml ofdistilled water. The solution of mesylate and water was cooled byimmersion in an ice bath. To the cooled solution was added 2.366 ml ofmercaptoethanol, which is 3 equivalent weights with respect to PEGhydroxyl groups. Also added was 16.86 ml of 2N NaOH base. The reactionwas refluxed for 3 hours, which means that the vapors rising from thereaction were continuously condensed and allowed to flow back into thereaction.

The poly(ethylene glycol) mercaptoethanol product was extracted threetimes with dichloromethane using approximately 25 ml of dichloromethaneeach time. The organic fractions were collected and dried over anhydrousmagnesium sulfate. The volume was reduced to 20 ml and the product wasprecipitated by addition to 150 ml of cold dry ether.

NMR analysis in d₆-DMSO dimethyl sulfoxide gave the following peaks forPEG—SCH₂CH₂OH: 2.57 ppm, triplet, —CH₂—S—; 2.65 ppm, triplet, —S—CH₂—;3.5 backbone singlet; and 4.76 ppm, triplet, —OH. The hydroxyl peak at4.76 ppm indicated 81% substitution. However, the 2.65 ppm peak for—S—CH₂— indicated 100% substitution. It has been observed that hydroxylpeaks frequently give low figures on percent substitution, and so the2.65 ppm peak for —S—CH₂— is considered to be more reliable and toconfirm 100% substitution.

Reaction 3. Reaction 3 represents peroxide oxidation of thepoly(ethylene glycol) mercaptoethanol product to convert the sulfur, S,to sulfone, SO₂. PEG ethanol sulfone is produced.

Twenty grams of PEG—SCH₂CH₂OH was dissolved in 30 ml of 0.123M tungsticacid solution and cooled in an ice bath. The tungstic acid solution wasprepared by dissolving the acid in sodium hydroxide solution of pH 11.5and then adjusting the pH to 5.6 with glacial acetic acid. Twenty ml ofdistilled water and 2.876 ml of 30% hydrogen peroxide, which has anequivalent weight of 2.5 with respect to hydroxyl groups, was added tothe solution of tungstic acid and poly(ethylene glycol) mercaptoethanoland the reaction was permitted to warm overnight to room temperature.

The oxidized product was extracted three times with dichloromethaneusing 25 ml of dichloromethane each time. The collected organicfractions were washed with dilute aqueous sodium bicarbonate and driedwith anhydrous magnesium sulfate. The volume was reduced to 20 ml. ThePEG ethanol sulfone product was precipitated by addition to cold dryethyl ether.

NMR analysis in d₆-DMSO dimethyl sulfoxide gave the following peaks forPEG—SO₂CH₂CH₂OH: 3.25 ppm, triplet, —CH₂—SO₂—; 3.37 ppm, triplet,—SO₂—3.50 ppm, backbone; 3.77 ppm, triplet, —CH₂OH; 5.04 ppm, triplet,—OH. The hydroxyl peak at 5.04 ppm indicated 85% substitution. However,the peak at 3.37 ppm for —SO₂—CH₂— indicated 100% substitution and isconsidered to be more reliable.

Reaction 4. Reaction 4 represents the final step in synthesis,isolation, and characterization of poly(ethylene glycol) chloroethylsulfone.

To synthesize the product, twenty grams of PEG—SO₂CH₂CH₂OH poly(ethyleneglycol) ethanol sulfone was dissolved in 100 ml of freshly distilledthionyl chloride and the solution was refluxed overnight. The thionylchloride had been distilled over quinoline. Excess thionyl chloride wasremoved by distillation. Fifty milliliters of toluene and 50 ml ofdichloromethane were added and removed by distillation.

To isolate the product, the PEG chloroethyl sulfone was dissolved in 20ml of dichloromethane and precipitated by addition to 100 ml of cold dryethyl ether. The precipitate was recrystallized from 50 ml of ethylacetate to isolate the product.

Nuclear magnetic resonance was used to characterize the product. NMRanalysis of PEG—SO₂CH₂CH₂Cl in d₆-DMSO dimethyl sulfoxide gave thefollowing peaks: 3.50 ppm, backbone; 3.64 ppm, triplet, —CH₂SO₂—; 3.80ppm, triplet, —SO₂—CH₂—. A small hydroxyl impurity triplet appeared at3.94 ppm. Calculation of the percentage substitution was difficult forthis spectrum because of the proximity of the important peaks to thevery large backbone peak.

Reaction 5. Reaction 5 represents conversion of poly(ethylene glycol)chloroethyl sulfone from reaction step 4 to poly(ethylene glycol) vinylsulfone and isolation and characterization of the vinyl sulfone product.

The PEG vinyl sulfone was readily prepared by dissolving solid PEGchloroethyl sulfone in dichloromethane solvent followed by addition oftwo equivalents of NaOH base. The solution was filtered to remove thebase and the solvent was evaporated to isolate the final productPEG—SO₂—CH═CH₂ PEG vinyl sulfone.

The PEG vinyl sulfone was characterized by NMR analysis in d₆-DMSOdimethyl sulfoxide. NMR analysis showed the following peaks: 3.50 ppm,backbone; 3.73 ppm, triplet, —CH₂—SO₂—; 6.21 ppm, triplet, ═CH₂; 6.97ppm, doublet of doublets, —SO₂—CH—. The 6.97 ppm peak for —SO₂—CH—indicated 84% substitution. The 6.21 ppm peak for ═CH₂ indicated 94%substitution. Titration with mercaptoethanol and 2,2′-dithiodipyridineindicated 95% substitution.

EXAMPLE 2

Thiol-selective Reactivity

Example 2 shows that PEG vinyl sulfone and its precursor PEG chloroethylsulfone are significantly more reactive with thiol groups (—SH) thanwith amino groups (—NH₂) or imino groups (—NH—). Compounds containingthiol groups are organic compounds that resemble alcohols, which containthe hydroxyl group —OH, except that in thiols, the oxygen of thehydroxyl group is replaced by sulfur. Thiols sometimes are also calledsulfhydryls or mercaptans. PEG vinyl sulfone contains the vinyl sulfonegroup —SO₂—CH═CH₂. PEG chloroethyl sulfone contains the chloroethylsulfone group —SO₂CH₂CH₂Cl.

Selectivity for thiols is important in protein modification because itmeans that cysteine units (containing —SH) will be modified inpreference to lysine units (containing —NH₂) and histidine units(containing —NH—). The selectivity of PEG vinyl sulfone for thiols meansthat PEG can be selectively attached to cysteine units, thus preservingprotein activity for specific proteins and controlling the number of PEGmolecules attached to the protein.

The relative reactivity of PEG vinyl sulfone with thiol and amino groupswas determined by measuring the rates of reaction of PEG vinyl sulfonewith N-α-acetyl lysine methyl ester and with mercaptoethanol. N-α-acetyllysine methyl ester is a lysine model containing an amino group and isabbreviated Lys—NH₂. Mercaptoethanol serves as a cysteine modelcontaining a thiol group and is abbreviated Cys-SH. Relative reactivityof PEG chloroethyl sulfone was similarly determined. This molecule mayserve as a “protected” form of the vinyl sulfone since it is stable inacid but converts to PEG vinyl sulfone upon addition of base.

Reactivity for PEG vinyl sulfone and for the PEG chloroethyl sulfoneprecursor was investigated at pH 8.0, pH 9.0, and at pH 9.5. Buffers forcontrolling the pH were 0.1 M phosphate at pH 8.0 and 0.1 M borate at pH9.0 and at pH 9.5. For measurement of mercaptoethanol reactivity, 5 mMethylenediamine tetraacetic acid (EDTA) was added to both buffers toretard conversion of thiol to disulfide.

For reaction of the PEG derivatives of the invention with Lys-NH₂, a 3mM solution of the PEG derivative was added under stirring to a 0.3 mMLys-NH₂ solution in the appropriate buffer for each of the three levelsof basic pH. The reaction was monitored by addition of fluorescamine tothe reaction solution to produce a fluorescent derivative from reactionwith remaining amino groups. The monitoring step was performed by adding50 μL of reaction mixture to 1.950 mL of phosphate buffer of ph 8.0followed by adding 1.0 mL of fluorescamine solution under vigorousstirring. The fluorescamine solution was 0.3 mg fluorescamine per ml ofacetone.

Fluorescence was measured 10 minutes after mixing. Excitation was shownat wavelength 390 nm. Light emission occurred at 475 nm. No reaction wasobserved in 24 hours for either PEG vinyl sulfone or PEG chloroethylsulfone at pH 8.0. At pH 9.5 the reaction was slow, but all amino groupswere reacted after several days.

For reaction of the PEG vinyl sulfone and PEG chloroethyl sulfoneprecursor with Cys-SH, a 2 mM solution of the PEG derivative was addedto a 0.2 mM solution of Cys-SH in the appropriate buffer for each of thethree levels of basic pH. The reaction was monitored by adding4-dithiopyridine to the reaction solution. The 4-dithiopyridine compoundreacts with Cys-SH to produce 4-thiopyridone, which absorbs ultravioletlight.

The monitoring step was performed by adding 50 μL of reaction mixture to0.950 mL of 0.1 M phosphate buffer at pH 8.0 and containing 5 mM EDTA,followed by adding one mL of 2 mM 4-dithiopyridine in the same buffer.

Absorbance of 4-thiopyridone was measured at 324 nm. Both PEG vinylsulfone and PEG chloroethyl sulfone showed reactivity toward Cys-SH,with PEG vinyl sulfone showing greater reactivity. At pH 9.0 thereaction is over within two minutes using the vinyl sulfone and within15 minutes using the chloroethyl sulfone. However, these reactions weretoo fast for determination of accurate rate constants. At pH 8.0 thereactions were slower, but still complete in one hour for vinyl sulfoneand in three hours for the chloroethyl sulfone. The conversion ofchloroethyl sulfone to vinyl sulfone is significantly slower than thereaction of vinyl sulfone with Cys-SH. Thus the rate of reaction forchloroethyl sulfone with Cys-SH appears to be dependent on the rate ofconversion of chloroethyl sulfone to vinyl sulfone. Nevertheless, thesereaction rates were still much faster than for the reaction withLys-NH2.

The above kinetic studies demonstrate the following points. PEG vinylsulfone is much more reactive with thiol groups than with amino groups,indicating that attachment of PEG vinyl sulfone to a protein containingboth cysteine and lysine groups proceeds primarily by reaction withcysteine. Since reactivity with amino groups is similar to imino groups,then reactivity of histidine subunits will also be much lower thanreactivity with cysteine subunits. Also, selectivity toward thiol groupsis accentuated at lower pH values for PEG chloroethyl sulfone and PEGvinyl sulfone, although the reactions of PEG chloroethyl sulfone aresomewhat slower.

The utility of many PEG derivatives is limited because they reactrapidly with water, thus interfering with attempts to attach thederivative to molecules and surfaces under aqueous conditions. Thefollowing Example 3 shows that PEG vinyl sulfone and PEG chloroethylsulfone are stable in water.

EXAMPLE 3

Hydrolytic Stability

PEG vinyl sulfone was dissolved in heavy water, D₂O deuterium oxide, andmonitored by NMR. Reaction did not occur. A solution of PEG chloroethylsulfone produced PEG vinyl sulfone in heavy water that was buffered withborate to pH 9.0. Monitoring with NMR showed that PEG vinyl sulfone,once produced, was stable for three days in heavy water.

PEG chloroethyl sulfone is stable in water until solution becomes basic,at which time it is converted into vinyl sulfone. Conversion to vinylsulfone has been demonstrated by dissolving PEG chloroethyl sulfone inwater at pH 7 and in borate buffer at pH 9. The PEG derivative isextracted into methylene chloride. Removal of methylene chloride,followed by NMR analysis showed that PEG chloroethyl sulfone is stableat a neutral pH of 7.0, and reacts with base to produce PEG vinylsulfone.

Vinyl sulfone is stable for several days in water, even at basic pH.Extensive hydrolytic stability and thiol-specific reactivity of PEGvinyl sulfone means that PEG vinyl sulfone and its precursor are usefulfor modification of molecules and surfaces under aqueous conditions, asshown in the following Example 4.

EXAMPLE 4

Protein Conjugation

Protein modification was demonstrated by attachment of the PEGderivative to bovine serum albumin (BSA) by two different methods. BSAis a protein. Native unmodified BSA contains cystine groups which do notcontain thiol groups. The cystine units are tied up as disulfidelinkages, S—S.

In the first method, m-PEG vinyl sulfone of molecular weight 5,000 wasreacted with unmodified BSA for 24 hours in a 0.1 M borate buffer at pH9.5 at room temperature. The solution contained 1 mg of BSA and 1 mg ofm-PEG vinyl sulfone of molecular weight 5,000 per ml of solution. Theresults from the Example 2 model compounds had indicated that lysinesubunits (and possibly histidine subunits) would be modified under theserelatively basic conditions and in the absence of free thiol groupsavailable for reaction.

Attachment to lysine subunits was demonstrated in two ways. First, sizeexclusion chromatography showed that the molecular weight of the proteinhad increased by approximately 50%, thus indicating attachment ofapproximately 10 PEGs to the protein. Second, fluorescamine analysisshowed that the number of lysine groups in the BSA molecule had beenreduced by approximately ten.

In the second method, the BSA was treated with tributylphosphine toreduce the disulfide S—S bonds to thiol groups, —SH, which are availablefor reaction. The modified BSA was then treated with PEG chloroethylsulfone at pH 8.0 in a 0.1 M phosphate buffer at room temperature for 1hour. The solution contained 1 mg of modified BSA and 1 mg of m-PEGchloroethyl sulfone of molecular weight 5,000 per ml of solution. Theresults showed that lysine groups were unreactive under theseconditions. However, thiol groups were reactive.

Attachment of the PEG to the protein was demonstrated by size exclusionchromatography, which showed an increase in the molecular weight of theprotein by about 25%. Fluorescamine analysis indicated no change innumber of lysine subunits in the protein, thus confirming that PEGattachment did not take place on lysine subunits. Substitution on thiolgroups was thereby confirmed.

The invention claimed herein has been described with respect toparticular exemplified embodiments. However, the foregoing descriptionis not intended to limit the invention to the exemplified embodiments,and the skilled artisan should recognize that variations can be madewithin the spirit and scope of the invention as described in theforegoing specification. On the contrary, the invention includes allalternatives, modifications, and equivalents that may be included withinthe true spirit and scope of the invention as defined by the appendedclaims.

1. A method for synthesizing a water soluble activated organic polymerhaving an active ethyl sulfone moiety wherein the linkage between thepolymer and the active ethyl sulfone moiety is stable againsthydrolysis, the method comprising the steps of covalently linking asulfur containing moiety directly to a terminal carbon atom of thepolymer and then converting the sulfur containing moiety to an activeethyl sulfone moiety having a reactive site located at the second carbonfrom the sulfone group, wherein said polymer is selected from the groupconsisting of poly(alkylene oxides), poly(oxyethylated polyols) andpoly(olefinic alcohols).
 2. The method of claim 1, further comprisingthe step of isolating the activated polymer having the active ethylsulfone moiety.
 3. The method of claim 1, wherein said step of linking asulfur containing moiety directly to a terminal carbon atom of thepolymer comprises the steps of activating at least one activatablehydroxyl moiety on the polymer and reacting the activated hydroxylmoiety with a compound comprising a thiol group covalently attached toan ethyl group to cause a sulfur atom to be covalently linked directlyto the terminal carbon of the polymer.
 4. The method of claim 3, whereinsaid step of activating at least one activatable hydroxyl moietycomprises hydroxyl substitution or replacement of the hydroxyl hydrogenwith a more reactive moiety.
 5. The method of claim 4, wherein said stepof activating at least one activatable hydroxyl moiety comprisesreplacing the hydroxyl group with a halide.
 6. The method of claim 4,wherein said step of activating at least one activatable hydroxyl moietycomprises reacting the hydroxyl moiety with an acid or acid derivativeto form a reactive ester.
 7. The method of claim 6, wherein the reactiveester is selected form the group consisting of sulfonate esters,carboxylate esters, and phosphate esters.
 8. The method of claim 6,wherein the acid or acid derivative is a sulfonyl acid halide selectedfrom the group consisting of methanesulfonyl chloride andp-toluenesulfonyl chloride.
 9. The method of claim 3, wherein saidreacting step comprises reacting the activated hydroxyl moiety withmercaptoethanol.
 10. The method of claim 1, wherein said converting stepcomprises oxidizing the sulfur atom to form an ethyl sulfone.
 11. Themethod of claim 10, wherein said oxidizing step comprises oxidizing thesulfur atom with an oxidizing agent selected from the group consistingof sodium perborate and hydrogen peroxide.
 12. The method of claim 10,wherein said converting step further comprises attaching a reactivegroup to the terminus of the ethyl sulfone moiety, thereby forming theactive ethyl sulfone.
 13. The method of claim 12, wherein said ethylsulfone comprises a terminal activatable hydroxyl group and said step ofattaching a reactive group to the terminus of the ethyl sulfone moietycomprises activating the terminal activatable hydroxyl moiety byhydroxyl substitution or replacement of the hydroxyl hydrogen with amore reactive moiety.
 14. The method of claim 1, wherein the polymer ispoly(ethylene glycol).
 15. The method of claim 1, wherein the polymer isa poly(ethylene glycol) molecule having at least one activatablehydroxyl group, and wherein said linking step comprises (a) activatingthe at least one activatable hydroxyl group by substituting the hydroxylgroup with a halide or replacing the hydroxyl hydrogen with an estergroup to form an ester or halide substituted poly(ethylene glycol)molecule, and (b) reacting the ester or halide substituted poly(ethyleneglycol) molecule with mercaptoethanol to substitute the mercaptoethanolradical for the ester or halide moiety; and further wherein saidconverting step comprises (c) reacting the mercaptoethanol substitutedpoly(ethylene glycol) molecule with an oxidizing agent to oxidize sulfurin the mercaptoethanol moiety to form an ethyl sulfone moiety and (d)converting the terminal hydroxyl group of the ethyl sulfone moiety to anester or halide moiety to form the active ethyl sulfone.
 16. The methodaccording to claim 15, further comprising reacting the active ethylsulfone moiety of the polymer with a thiol-containing surface orbiologically active molecule.
 17. The method according to claim 16,wherein the active ethyl sulfone moiety of the polymer is reacted with athiol-containing protein or fragment thereof.
 18. The method accordingto claim 1, further comprising reacting the active ethyl sulfone moietyof the polymer with a thiol-containing surface or biologically activemolecule.
 19. The method according to claim 18, wherein the active ethylsulfone moiety of the polymer is reacted with a thiol-containing proteinor fragment thereof.
 20. The method according to claim 1, wherein thepolymer comprising the active ethyl sulfone moiety is a homobifunctionalpolymer or a heterobifunctional polymer.